Project Summary: Hemorrhagic fever (HF) caused by Lassa virus (LASV) represents an important public health problem in its endemic regions of West Africa, and LASV poses also a credible biodefense threat. On the other hand, mounting evidence indicates that the worldwide-distributed arenavirus LCMV is a neglected human pathogen. of clinical significance. No FDA-licensed arenavirus vaccines exist and current anti-arenaviral therapy is limited to an off label use of ribavirin (Rib) that is only partially effective. Therefore, there is an unmet need for novel anti-arenaviral therapeutics. We hypothesize that this task can be facilitated by identifying inhibitors of the arenavirus ribonucleoprotein (vRNP) that directs virus RNA replication and gene transcription. The use of live LASV to identify candidate antiviral drugs is complicated by the requirement of BSL4 containment. To overcome this obstacle we have developed cell lines expressing LASV and LCMV functional vRNP where expression of reporter genes serve as accurate surrogates of vRNP activity. We propose to use these vRNP- expressing cell lines to develop virus free, cell-based assays suitable for HTS to identify inhibitors of LASV and LCMV vRNPs. For this, we will complete the following aims: Aim 1. To develop cell-based assays suitable for HTS to identify inhibitors of the activity of LASV and LCMV vRNP. We will use LASV and LCMV vRNP-expressing cell lines to develop cell-based assays (96-well format) suitable for HTS. Assay parameters will be optimized to be compatible with HTS: S/B ? 10, CV ? 10% and Z?- values > 0.5 based on Max and Min reporter gene signals corresponding to 0 and 250 M Rib, respectively. Assay miniaturization studies will be implemented to develop a 384-well format. Aim 2. To develop counter screen and secondary assays for hit validation. We will develop secondary assays, including cell-based infection with live LASV and LCMV to authenticate hits and rule out false positives including those related to toxicity, effects on Pol-II activity and interference with reporter gene activity. Aim 3. To perform a pilot screen using LASV and LCMV vRNP cell-based assays. We will work with the TSRI MSC to use the optimized 384-well format assays in pilot screens using the LOPAC, NIH Clinical Collection I and II, and Maybridge Diversity Set version 4 libraries. In addition, we will screen the high value Gilead nucleoside compound collection that has led to the discovery of inhibitors of viral polymerases that were developed into marketed antiviral drugs against HCV and HIV. Threshold levels for primary hit identification will be: ability to inhibit (? 50%) vRNP activity and lack of significant toxicity (> 70% cell viability). Candidate hits will be confirmed using the secondary and counter screen assays described in aim 2.